The mouse mammary tumor virus genome (MMTV) is of particular relevance to the study of gene regulation because its transcription is regulated by an interaction with a hormone-receptor complex. We have begun to analyze the nucleotide signals responsible for this regulation in two types of experiments. In short-term assays, the MMTV long terminal repeat sequences (LTRs) were coupled to the prokaryotic gene, chloramphenicol-acetyltransferase (CAT), which is a sensitive and accurate indicator of gene expression. Intact LTR sequences or deletion mutants thereof were introduced into tissue culture cells in the presence or absence of dexamethasone in an attempt to decipher those sequences responsible for hormone responsive regulation. In a second type of assay, based on transformation using similar constructs which had the transforming Harvey rat gene (p21) in the position analogous to that of CAT, we determined the transformation efficiency of the constructs harboring various mutants of these regulatory signals, in the presence or absence of steroid hormone. In general, the results of these two types of assays were in good agreement. Deletion of sequences from the 5' end of the LTR resulted in a general decrease in gene activity in the presence of the hormone, but an increase in activity in the absence of hormone. One possibility under investigation is that negative regulatory sequences suppress gene expression in the absence of the specific inducer (dexamethasone). In a parallel set of studies, we have confirmed data from other laboratories, indicating that the upstream control signals of MMTV can regulate the activity of other heterologous promoters in a hormone-dependent fashion.